一株感染家蚕的高致病性白僵菌的筛选及复合诱变

【背景】白僵蚕中的生物活性物质在医疗、保健品及化妆品行业有着广泛的应用。目前,许多人工养殖僵蚕基地在实际生产中使用的菌种多为未进行纯化优选的自然感病死亡僵蚕孢子粉且无固定的施用浓度,使得蚕的僵化死亡率难以保证。提高白僵菌菌株的致病力并筛选性状优良的高毒力菌株是工厂化生产白僵蚕研发的重要方向。【目的】利用紫外-微波复合诱变技术筛选高毒力菌株,为僵蚕工厂化生产提供优良菌株。【方法】利用孢子稀释法从山西省养殖农户中自然感染白僵菌的家蚕中分离获得一株原始白僵菌,运用紫外-微波复合的方式对该菌株进行诱变,并比较诱变前后菌株的产孢量及对家蚕的致病力。【结果】分离得到的原始菌株经鉴定为球孢白僵菌(Beauveria bassiana),命名为Beauveria bassiana Bb1003。通过对致死率和正突变率的考察,确定紫外-微波复合诱变的最佳诱变条件为紫外(功率为15 W)照射30 min,微波(功率为800 W、额定微波频率2 450 MHz)辐照60 s。筛选后得到6株复合诱变菌株(UMCM1、UMCM2、UMCM3、UMCM4、UMCM5和UMCM6)。菌株UMCM2对家蚕的僵化率高达...     

Evaluation of the pathogenicity and infectivity of entomopathogenic hypocrealean fungi,isolated from wild mosquitoes in Japan and Burkina Faso,against female adult Anopheles stephensi mosquitoes

We investigated the potential of entomopathogenic hypocrealean fungi that naturally occur in or on adult mosquitoes for use as biocontrol agents of vector mosquitoes. The fungi were isolated from wild mosquitoes collected in Japan and Burkina Faso using two isolation methods (with and without surface sterilization). Detected fungal species included Beauveria bassiana sensu lato, Isaria spp., Paecilomyces spp., Lecanicillium spp., and Simplicillium spp. These isolates were used in bioassays against adult female Anopheles stephensi mosquitoes. The median survival time ranged from 5.8 to 14.9 d (control, 17.0 d). Reduced survival times were observed in the isolates from surface-sterilized mosquitoes from Japan, with the isolate B. bassiana s.l. 60-2 exhibiting the highest virulence. This study indicates that adult mosquitoes are naturally infected with various entomopathogenic hypocrealean fungi, and that some of these isolates have the potential for use as fungal pesticides to control vector mosquitoes.     

Isolation and characterisation of Beauveria bassiana isolates from phylloplanes of hedgerow vegetation

A leaf imprinting technique combined with a selective medium was used to document the natural occurrence of Beauveria bassiana on phylloplanes of typical hedgerow plants (grasses, stinging nettle and hawthorn) in May, July and September in a hedgerow in Denmark. The density of B. bassiana (as measured by numbers of colony forming units) was greatest in September and on lower nettle leaves. B. bassiana was isolated from phylloplanes in a different hedgerow the following year and a similar picture of occurrence was found. Genetic diversity of selected in vitro isolates were characterised by Universally Primed (UP) PCR, and 13 distinguishable banding patterns were found at the two localities. Of these, four were shared between the field sites and all plant species harboured isolates of B. bassiana with at least two different banding patterns. The isolation method described represents a valuable tool for studying naturally occurring B. bassiana and for rapid isolation of indigenous strains of the fungus for future development of biocontrol agents. The significance of the findings for the life-cycle of B. bassiana is discussed.     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.     

A population survey of Beauveria bassiana in the microhabitat of the red turpentine beetle,Dendroctonus valens,in Chinese pine forests

Field survey of the entomopathogenic fungus Beauveria bassiana in association with the red turpentine beetle, Dendroctonus valens, was undertaken in three pine plantations in Northern China. In total, 88 strains of B. bassiana sensu lato were isolated from the soil, bark, beetle frass, living adult and cadaver samples and soil was proved to be an important inoculum reservoir for fungal entomopathogens. Of these, 77 isolates were included for genetic diversity analysis by PCR for inter-simple sequence repeats (ISSR). Genetic diversity and population structure analysis of the isolates from three sites and five niches demonstrated high genetic diversity and heterogeneity between and/or within populations. Wright's statistics revealed a high gene flow rate (4.529) among the three populations, especially among the soil-derived isolate subpopulations. Low variation was mainly caused (94.8%) by variation among different substrates, suggesting the importance of microhabitat substrates on genetic diversity of B. bassiana. Phylogenetic variation was not associated with geographic distance.     

Isolation of Beauveria species from Lebanon and evaluation of its efficacy against the cedar web-spinning sawfly, Cephalcia tannourinensis

Larvae of the cedar web-spinning sawfly, Cephalcia tannourinensis Chevin (Hymenoptera: Pamphiliidae), infected with a white fungus were collected from the Tannourine-Hadath El-Jebbeh cedar forest. Macro- and micro-morphological data based on the examination of colonies, conidiophores, and conidial shape of the fungus suggested a Beauveria species. Sequence analysis of the internal transcribed spacer regions of the isolated fungus showed that it is most closely related to isolates of B. bassiana Clade C. The present study showed that the isolated B. bassiana is a naturally occurring entomopathogenic fungus parasitizing the larvae of C. tannourinensis in Lebanon. Laboratory bioassays showed that B. bassiana caused high mortality of eggs and larvae. The infected eggs turned brownish in color, while larvae of the first instar ceased feeding and showed immobility and rigidity within 5 days and before sporulating conidial mat appeared on their cuticle. Second and third larval instars took longer time to show fungal sporulation: mortalities ranged between 85 and 100% within 7 days when treated with different conidial concentrations. The efficacy of control of C. tannourinensis using B. bassiana was higher or equal to the reference insect growth regulator, diflubenzuron, suggesting the possibility of its success as a biological control agent.     

Niche separation of species of entomopathogenic fungi within the genera Metarhizium and Beauveria in different cropping systems in Mexico

Entomopathogenic fungi from the genera Beauveria and Metarhizium, were isolated from soil using the Galleria mellonella baiting method, and from infected white grub larvae from a diversity of cropping systems in Puebla and Guanajuato, Mexico. Isolates were identified to species level using Bloc and Elongation Factor 1-α sequence information. Although widespread, Beauveria bassiana (41 isolates) was only isolated from soil and not from infected white grubs. In contrast, Beauveria pseudobassiana (six isolates) was predominantly isolated from white grub larvae (only one isolate from soil). Haplotype analysis of B. bassiana Bloc sequences identified 25 haplotypes indicating substantial genetic diversity; neither geographical origin nor crop type explained this genetic variation. Metarhizium brunneum (three isolates) and Metarhizium robertsii (17 isolates) were also only isolated from soil, while Metarhizium anisopliae (six isolates) and Metarhizium pingshaense (four isolates) were only isolated from white grub larvae. M. anisopliae was only found infecting Paranomala species while M. pingshaense was only found infecting Phyllophaga species. Species diversity in Metarhizium was influenced by crop type. Our results showed that entomopathogenic fungi species could co-exist in the same soil ecosystem but in separate niches. The potential ecological roles of these species are discussed.     

The molecular diversity of different isolates of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Bals.) Vuill. as assessed using intermicrosatellites (ISSR<Subscript>s</Subscript>)

Inter-microsatellite PCR (ISSR-PCR) markers were used to identify and to examine the genetic diversity of eleven Beauveria bassiana isolates with different geographic origins. The variability and the phylogenetic relationships between the eleven strains were analyzed using 172 ISSR-PCR markers. A high level of polymorphism (near 80%) was found using these molecular markers. Seven different isolates showed exclusive bands, and ISSR primer 873 was able to distinguish between all the strains. The dendrogram obtained with these markers is robust and in agreement with the geographical origins of the strains. All the isolates from the Caribbean region were grouped together in a cluster, while the other isolates grouped in the other cluster. The similarity exhibited between the two clusters was less than 50%. This value of homology shows the high genetic variability detected between the isolates from the Caribbean region and the other isolates. ISSR-PCR markers provide a quick, reliable and highly informative system for DNA fingerprinting, and allowed the identification of the different B. bassiana isolates studied.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

Hypocrealean fungi associated with populations of <Emphasis Type="Italic">Ips typographus</Emphasis> in West Carpathians and selection of local <Emphasis Type="Italic">Beauveria</Emphasis> strains for effective bark beetle control

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC₅₀ ranging from 0.72 to 2.05?×?10⁶ conidia ml^?1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.     

Molecular tracing of white muscardine of the silkworm,Bombyx mori I: genetic structure analysis of Beauveria bassiana populations as the causal agents in China

Beauveria bassiana is a ubiquitous entomopathogenic fungus, which causes white muscardine in the silkworm, Bombyx mori, and is an important limiting factor in global silk production. Knowledge of the genetic structure of B. bassiana populations as the causal agents in China will aid in tracing the origins of muscardine and in developing effective control strategies. In the present study, a total of 489 B. bassiana isolates were obtained from silkworm cadavers in 12 different provinces of China. An analysis of the genetic structure of the causal agent populations using the internal simple sequence repeat technique to trace their origins revealed a high level (100%) of DNA polymorphism. An unweighted pair group method with arithmetic mean dendrogram revealed that the 489 B. bassiana isolates were heterogenic and polyphyletic and displayed typical regional distribution, although the genetic differentiations were not associated with geographic distance. The genetic differentiation (G_st) among populations reached 0.406, with the maximum G_st between populations reaching 0.647. The general gene flow (N_m) was low at 0.366, with the lowest at only 0.137, suggesting that the low gene flow maintained a high degree of differentiation. These data indicated that the white muscardine in each local silkworm population was predominantly caused by a native group of B. bassiana, rather than by any exotic strain as in the case of biological control based on B. bassiana insecticides.     

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

Preliminary study of genetic variation in Hawaiian isolates of Beauveria bassiana [Hypocreales, Cordycipitaceae

There have been no previous surveys documenting genetic diversity in Beauveria bassiana (Balsamo) Vuillemin in Hawaii. We used PCR primers and DNA sequencing to genetically characterize 14 isolates of B. bassiana collected from insects in east Hawaii island (the largest Hawaiian island, known as the ‘Big Island’) and compared these with the ‘GHA’ strain found in the commercial product BotaniGard®. Twelve of the 14 Hawaiian isolates were unique and the GHA strain was not among those isolated from the wild. Our data provides evidence that genetic diversity of B. bassiana in Hawaii is high over small spatial scales.     

Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system

Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and β-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.     

Isolation and characterization of Beauveria spp. associated with exotic bark beetles in New Zealand Pinus radiata plantation forests

A survey of entomopathogenic fungi associated with Hylastes ater and Hylurgus ligniperda was undertaken in six Pinus radiata forest sites in New Zealand. In each site soil, bark and frass associated with beetle galleries were sampled, as well as a selection of live beetles, larvae and mycosed cadavers. The density of Beauveria spp. (as measured by colony forming units) varied both within and between the sites and substrates sampled. The highest CFU density detected was 4 × 10⁶ CFU/g dry frass, which was collected from a stump at the Mahurangi site in the North Island. A number of isolates were selected and characterized using PCR amplification of a terminal region of the EF1-α fragment. This analysis revealed that two Beauveria species, Beauveria caledonica and Beauveria bassiana, were commonly associated with bark beetle habitat. B. caledonica was the only species isolated from mycosed beetles, and was also recovered from soils, bark and frass, though not live insects. B. bassiana was also found in soils, bark, frass and live insects; however, it was not isolated from mycosed cadavers in this study. It is speculated that B. caledonica is the common ‘natural’ pathogen of bark beetles in their breeding habitat. A third species, Beauveria malawiensis, was isolated once from soil. This is the first report of this species from New Zealand. Thirteen isolates (selected from all substrates sampled and live beetles) were selected for laboratory bioassays. Under laboratory conditions all isolates were pathogenic to H. ater and H. ligniperda adults; there was little difference among the isolates in terms of efficacy despite the paucity of B. bassiana infection observed in the field. The mechanisms behind the lack of field infection observed for B. bassiana are not known, but are likely to be linked to limiting environmental factors in the breeding sites. These results indicate potential risks associated with the selection of strains for use in biological control programmes solely on the basis of laboratory bioassays, especially if the mode of action and biology of the species used are not fully understood. The importance of selecting strains which are environmentally competent is evident. Overall, however, this study demonstrates that entomopathogenic fungi can play a significant role in the regulation of H. ater and H. ligniperda populations in pine plantations.     

Genetic diversity among summer and winter Beauveria bassiana populations as revealed by AFLP analysis

The genetic diversity of Beauveria bassiana was investigated by comparing 40 isolates collected from summer and overwintering populations of Sunn pest from different areas in Syria and Turkey, using amplified fragment length polymorphism (AFLP) markers. Considerable genetic variability among B. bassiana isolates was revealed. The examined isolates were divided into three distinct clusters (A, B, and C). Within these clusters, the summer isolates from Syria and Turkey were grouped together in three sub-clusters (A3, A4, and B2). Also, principal coordinate analyses (PCA) showed clear separation (62.5%) between summer and winter isolates. These differences in the genetic structure may be explained by the variety of eco-geography over the sampled areas of B. bassiana isolates. This information on genetic variation among summer and winter B. bassiana isolates is helpful in designing an effective integrated pest management program for Sunn pest.     

Aspergillus bombycis genotypes (RFLP) from silkworm cultivation

 Eighteen isolates of Aspergillus bombycis from samples of dust, insect frass, and soil collected from eight silkworm rearing facilities in Japan, as well as single silkworm rearing facilities in Indonesia and Malaysia, were subjected to DNA fingerprinting. PstI digests of total genomic DNA from each isolate were probed using the pAF 28 repetitive sequence. Among 18 isolates analyzed, 7 distinct DNA fingerprint groups were identified, including GTAb-2 isolated from rearing facilities in four prefectures of Japan. Aspergillus bombycis isolates share several features in common with domesticated yellow-green aspergilli, the koji molds used in traditional Oriental food fermentations, including a loose and deep colony texture, smooth-walled stipes, and the absence of sclerotia. Although aflatoxin is unknown from koji molds, all isolates of A. bombycis produced B and G aflatoxins. Aflatoxin has been linked to increased virulence in Aspergillus disease of silkworms, and there should be strong selection for aflatoxin production among clonal populations of A. bombycis adapted to silkworm cultivation. A hypothesis is offered that A. bombycis isolates from silkworm cultivation represent highly adapted forms of yet to be discovered “wild” populations that may infect Bombyx mandarina. Received: December 24, 2002 / Accepted: March 10, 2003 RID="*" ID="*" Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the products, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable Acknowledgments We thank Dr. Akira Nakagiri, Institute for Fermentation, Osaka, for providing Aspergillus cultures isolated from diseased silkworms.     

Detection and characterization of Wolbachia infection in silkworm

Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects.